Non-Toxic immunogens derived from a retroviral regulatory protein, antibodies, preparation method therefor, and pharmaceutical compositions containing same

ABSTRACT

A non-toxic immunogenic compound, which may be administered to humans, is derived from an HIV-1, HIV-2, HTLV-1 or HTLV-2 viral regulatory protein by chemical processing using a coupling agent such as an aldehyde, or from a carrier protein activated by pre-processing using an aldehyde. This compound is capable of being recognized by antibodies to the viral regulatory protein and retains sufficient immunogenic properties to produce antibodies that neutralize or block the native protein, while losing at least 50% of the toxic biological properties of the native protein.

The present invention relates to new retroviral immunogens which useretroviral regulatory proteins, the essential biological properties ofwhich will have been inactivated beforehand such that their immunogenicproperties are retained or increased, a process for their preparationand pharmaceutical compositions comprising them. These new "inactivated"immunogens can be used to induce active immunization in humans which iscapable of preventing or correcting the deregulatory effects which thenative proteins from which they are produced may help to produce.

The present invention also relates to new antibodies obtained by usingthese "inactivated" immunogens, processes for their preparation andpharmaceutical compositions for passive immunization which comprisethem.

The Tat molecule is an HIV regulatory protein which is not found in theviral particle but is coded by the HIV-1 genome. Inside infected cells,this protein coded by the proviral DNA plays a transactivating role onviral or cell genes. However, this genetic regulatory protein can alsobe found outside cells in the circulating extracellular medium, excretedwithin the debris of dead cells in the native or fragmented state or bya secretion process. Its presence in the circulating medium explains theexistence of anti-Tat antibodies detectable in some seropositivesubjects. In this extracellular context of Tat, the terminal C segmentwhich carries the RGD residues recognized by the integrins of the cellsurface and the base region of the molecule (residues 45-70) enable it,as do bacterial toxins, to act on the non-infected cells of differenttissues. The circulating Tat protein thus acts like a true viral toxin,can exert harmful effects on endothelial cells and, in combination withthe growth factor BFGF, can contribute to the neoangiogenesis ofKaposi's sarcoma, which characterizes the AIDS disease. The circulatingTat protein can also aggravate the immunosuppression which becomesestablished progressively with AIDS, either by direct immunosuppressiveaction on the T cells or by helping to deregulate the production ofinterferon-α by the cells presenting the antigen, called APC(macrophages or dendrite cells).

Acquired immunodeficiency syndrome (AIDS) is defined clinically byopportunistic diseases due to immunosuppression or by Kaposi's sarcoma.Acquired immunosuppression, established progressively in the course ofHIV infection, manifests itself biologically by the loss ofimmunological reactivity of T cells (cytostasis and the reduction in theproduction of IL2), after stimulation in the first instance by memoryantigens, and then by alloantigens, and finally by mitogens (PHA). Thisimmunosuppression is associated with excessive production ofinterferon-α and -γ.

The cytopathogenic mechanisms which induce immunosuppression are complexand involve various factors of viral origin, which act either directlyon the immunity cells, T lymphocytes and APC, or indirectly via thecytokine system. The envelope protein gp120 which is carried by theHIV-1 particle and of which the extracellular presence is measured bythe serum viral charge can thus induce anergy of T cells of phenotypeCD4 directly. On their part, the HIV regulatory proteins, in particularTat in its extracellular configuration of circulating viral toxin, alsoseem to induce a direct immunosuppression of T cells or other pathogeniceffects, for example by the fact that in vitro T cells activated by amemory antigen or by anti-CD3 antibodies no longer proliferate in thepresence of the Tat molecule. In addition, the circulating Tat proteinseems to facilitate excessive production by the APC of interferon-α, acytostatic and apoptogenic cytokine which is capable of amplifying theimmunosuppresion and apoptosis observed with the AIDS disease. In fact,the secretion of interferon-α by macrophages no longer seems halted inthe presence of Tat by a retroregulation (feedback), which in the normalstate controls this production of interferon-α (refractory period).

Kaposi's sarcoma manifests itself clinically by the appearance ofvascular nodules representing neoangiogenesis starting from endothelialcells. These cells, which have been activated by inflammatory processesgenerating the production of cytokines (interferon-γ, IL1 and IL6),produce BFGF (basic fibroblastic growth factor) and multiply. Theproliferation of activated endothelial cells in vitro is increased bythe presence in the medium of the Tat protein. The anti-Tat antibodiesblock the proliferative effects of the Tat protein in vitro. The actionof Tat on the endothelial cells which carry adhesin from the family ofintegrins on their surface is explained by the presence of the RGDsequence recognized by these molecules.

The neoangiogenesis which underlies Kaposi's sarcoma in vivo was thussaid to be promoted by the Tat regulatory protein in its extracellularconfiguration, which could be recognized, thanks to its terminal Cfragment containing the RGD sequence, by the integrins of endothelialcells. Furthermore, the Tat may also act by its base region rich inresidues K and R, and consequently be capable of bonding to heparinsulphate of the extracellular matrix, which concentrates the growthfactor BFGF. The proliferation of endothelial cells induced by thegrowth factors is thus increased by the presence of Tat and generatesneoangiogenesis. This effect on the growth of endothelial cells isreduced in vitro by the action of anti-Tat antibodies.

It thus appears desirable to block the harmful activity of retroviralregulatory proteins, in particular of Tat circulating in theextracellular media (blood, lymph, interstitial media . . . ), the trueviral toxins.

As regards the HIV viruses, up to the present time attempts atvaccination have been made with the aid of structure proteins orfragments of structure proteins of these viruses, but never with the aidof regulatory proteins or fragments of regulatory proteins of theseviruses.

Thus, it has been found, surprisingly, that like the bacterial toxins(tetanic, diphtheric or botulic), the toxic activity of which isneutralized by specific antibodies, the harmful effects of regulatoryviral proteins, and in particular of Tat, a true toxin of HIV in itsextracellular configuration--which these exert by immunosuppression of Tcells, by deregulation of the production of interferon-α by the APC orby the neoangiogenesis which underlies Kaposi's sarcoma--are abolishedin the presence of specific anti-Tat antibodies, as will be seen belowin the experimental part.

The same applies to other regulatory proteins of viruses such as HIV-1,HIV-2, HTLV-1 or HTLV-2. It would thus be desirable to have availableimmunogens which can be administered to humans and are capable ofproducing such antibodies, and also to have available such antibodies,for both curative and preventive purposes. In fact, the compounds of theprior art used as immunogens may be toxic to humans (see, for example,WO-A-9118454), in particular those containing base regions. These arenon-modified native fragments of Tat, Nef or Rev, in contrast to thepresent invention, which is based on the inactivation of these proteinsor protein fragments.

The present Application thus relates to immunogenic compounds which canbe administered to humans, since they are non-toxic, characterized inthat they are derived from an HIV-1, HIV-2, HTLV-1 or HTLV-2 virusregulatory protein by chemical treatment with the aid of a couplingagent such as an aldehyde, or from a carrier protein activated bypretreatment with the aid of an aldehyde, preferably formaldehyde orglutaraldehyde, enabling them to be recognized by antibodies to the saidregulatory protein, and to retain sufficient immunogenic properties tocreate antibodies which neutralize or block the said native protein,while having lost at least 50%, in particular at least 80%, moreparticularly 95%, of the toxic biological properties of the said nativeprotein.

These compounds, by analogy to bacterial toxoids such as tetanus toxoid,will be qualified below as "toxoids".

In fact, like the conventional bacterial toxoids, they are devoid ofproper toxicity, but are nevertheless capable of causing immunization byadministration to a subject.

The chemical treatment below can be supplemented by a physicaltreatment, such as, for example, irradiation, and especially UVirradiation, with the aim of reducing the residual toxicity of theimmunogenic compounds according to the invention.

The above toxoids can be prepared, for example, from a peptide which hasa sequence identical or similar to a peptide sequence of a regulatoryprotein, such as Tat, and can be obtained, for example, by conventionalpeptide synthesis on resin or by genetic engineering. All theseprocesses are well-known in the prior art.

In order to check that the modified regulatory protein or its modifiedfragment according to the invention is recognized readily by antibodiesto the said native regulatory protein, the formation of antigen-antibodycomplexes, for example, can be checked immunologically by ELISA in thepresence of specific antibodies, as will be seen below in theexperimental part.

In order to determine whether the immunogenic properties of theregulatory protein have been retained sufficiently to create antibodieswhich neutralize the said native protein, mammals (rabbits, rats, mice)can be immunized, for example, with the aid of an immunogenic compoundaccording to the invention and it can be checked that the antibodiesproduced neutralize the toxic activities of the regulatory protein, aswill be seen for Tat in the experimental part.

In order to determine whether the modified regulatory protein has lostat least 50% of its toxic biological properties, it is possible tostudy, for example, the effect of the regulatory protein, such asinactivated Tat, on the immunosuppression of T cells or on theproduction of interferon-α by activated mononuclear cells of peripheralblood, or also on the neoangiogenesis induced by the regulatory protein.

The inactivation of the Tat regulatory protein is checked, for example,by "Tat rescue assay", using a Tat-deficient non-infectious HIV mutantcultured on the cell line HLM-1, replication of which depends on anexogenous supply of native Tat.

The immunogenic compound can be derived from any of the HIV-1, HIV-2,HTLV-1 or HTLV-2 virus regulatory proteins, and in particular Vif, Rev,Nef and Tat of the HIV-1 and HIV-2 viruses; Rev, Nef and Tat are used inparticular, more particularly Rev and Tat, and preferably the latter.

The Tax protein of HTLV-1 or HTLV-2 may also be mentioned.

There may also be mentioned especially the regions outside the baseregions of Tat and Rev, that is to say outside regions 49 to 57 of Tatand 35 to 50 of Rev, or overlapping these regions by at most 4 aminoacids, preferably at most 2 amino acids, in particular the peptide HisGln Val Ser Leu Ser Lys Gln Pro Thr Ser Gln Pro Arg Gly Asp (SEQ IDNO:1).

"Derived from" or "to derive" an HIV1, HIV2, HTLV1 or HTLV2 virusregulatory protein is understood as meaning that the immunogeniccompound can be made up of all or a fragment of the regulatory proteinand may include one or more modifications in the amino acids of thisprotein or fragment, such as deletions, substitutions, additions orfunctionalizations, such as acylation of amino acids, to the extent thatthese modifications remain within the context specified above (absenceof toxicity, immunological characteristics). For example, in general,the replacement of a leucine residue by an isoleucine residue does notmodify such properties; the modifications should generally concern lessthan 30% of the amino acids, preferably less than 20%, and especiallyless than 10% over the homologous segments of the regulatory protein ofat least 8 amino acids, in particular at least 12 amino acids. Afragment may comprise 8 to 60 amino acids, for example, preferably 12 to40 amino acids and in particular 25 to 40 amino acids. Residues 65-80 inthe C terminal of the Tat of HIV-1 may be mentioned by way of example.

Under preferential conditions, the immunogenic compounds of theinvention comprise at least 50% of the total regulatory protein,preferably at least 70%, in particular at least 90%, and especially allor almost all the said protein.

Generally, as regards the modifications, homology or similarity betweenthe modified immunogen and the native protein or part of native protein,as well as the dimensions of the immunogenic compound, and furthermorethe methods of use or of coupling of the immunogenic compound accordingto the invention to an immunogenic protein, such as the tetanic toxoid,reference may be made in particular to WO-A-86/06 414 or toEP-A-0.220.273 or also to PCT/US.86/00831, which are equivalents, thedisclosure of which is incorporated here by reference.

The immunogenic compounds according to the invention can be used asfollows:

An immunogenic compound according to the present invention isadministered to a patient, for example subcutaneously orintramuscularly, in an amount sufficient to be effective at thetherapeutic level, in a subject having need of such a treatment. Thedose administered can range, for example, from 100 to 1,000 μgsubcutaneously, once a month for three months, and then periodicallyaccording to the level of serum antibodies induced, for example every2-6 months.

A composition according to the invention can be administered by anyconventional route customary in the field of vaccines, in particularsubcutaneously, intramuscularly, intravenously or orally. Theadministration can take place in a single dose or be repeated once orseveral times after a certain interval of time.

The present Application thus also relates to a curative or preventivepharmaceutical composition, characterized in that it comprises animmunogenic compound as defined above as the active principle. Theimmunogenic compound can be formulated by itself or mixed with apharmaceutically acceptable excipient, such as an adjuvant.

The invention also relates to medicaments, characterized in that theycomprise immunogenic compounds as defined above, that is to say theabove immunogenic compounds, for their use in a method for therapeutictreatment of the human or animal body, and to the use of such animmunogenic compound for the preparation of a curative or preventivemedicament intended for treatment or prevention of the harmful effectsof the above regulatory proteins, and in particular Tat. In fact, thecompounds according to the invention have lost their toxic propertiesand can therefore be administered to humans, as will be seen below inthe experimental part.

The administration of immunogenic compounds according to the inventioncorresponds to an active immunotherapy. It may also be of interest tocarry out a passive immunotherapy, that is to say to provide a patientdirectly with the antibodies which he requires to neutralize the harmfuleffects of HIV-1, HIV-2, HTLV-1 or HTLV-2 virus regulatory proteins.

These anti-regulatory protein antibodies can be obtained conventionallyand, by way of example, after immunization of a mammal, human or animal,with the aid of an immunogenic compound as defined above, by cloninghuman B lymphocytes transformed by the Epstein Barr virus and thencollection of the required antibodies secreted by the said transformed Blymphocytes, or also by genetic recombination from a library of phages.

The present Application thus also relates to such processes for thepreparation of anti-HIV-1, -HIV-2, -HTLV-1 or -HTLV-2 virus regulatoryprotein antibodies, and in particular anti-Tat toxoid antibodies, and inparticular a process for the preparation of an above antibody,characterized in that a mammal is immunized with the aid of animmunogenic compound as defined above.

The present Application also relates to an anti-HIV-1, -HIV-2, -HTLV-1or -HTLV-2 virus regulatory protein antibody, and in particularpolyclonal or monoclonal antibodies obtained from human or mammaliansubjects immunized by carrying out the processes described above.

These specific antibodies may originate:

1. either from the subject himself, induced by an active immunization(vaccination) with a biologically inactivated but immunogenic regulatoryprotein, in particular Tat. Such an immunogenic compound in the case of,for example, Tat is called Tat-toxoid, by analogy to bacterial toxoids,

2. or from a foreign allo- or xenogenic organism, administered to thesubject by passive immunization (serotherapy). Allogenic antibodies (inhumans) could be generated in volunteer non-infected subjects afteractive immunization (vaccination) with an immunogenic protein or itsderivatives according to the invention, in particular Tat (Tat-toxoid orpeptide fragments of Tat according to the invention). These passivelyadministered antibodies, whether allogenic (human) or xenogenic(animal), could be complete monoclonal or polyclonal antibodies orfragments F(ab')2 or Fab of the antibody.

"Anti-regulatory protein antibody" is understood as meaning monoclonalor polyclonal antibodies or fragments F(ab')2 or Fab of theseantibodies, or also anti-regulatory protein antibodies obtained bygenetic construction from a library of phages.

The allogenic antibodies of human origin are:

either polyclonal--which can be obtained in volunteer seronegativesubjects immunized with an immunogenic compound according to theinvention, in particular Tat-toxoid or peptide fragments of Tat.

or monoclonal, from specific clones of B cells transformed by the EBVvirus of these immunized individuals (EBV-specific B lines).

The xenogenic antibodies originate from animals hyperimmunized with animmunogenic compound according to the invention, in particular Tat orits derivatives (Tat-toxoid, detoxified peptide fragments of Tataccording to the invention), and are

either polyclonal, originating from hyperimmunized animals,

or monoclonal, obtained after hybridization according to the Kohler andMilstein technique on splenic cells or adenocytes with a myelomatousline, type x63, in particular x63AG3. In this case, horse or rabbitantibodies are preferred.

The present Application also relates to a process for the preparation ofanti-HIV-1, -HIV-2, -HTLV-1 or -HTLV-2 virus regulatory proteinantibodies, characterized in that a mammal, human or animal, isimmunized with an immunogenic compound as defined above.

The present invention also relates to a process for the preparation ofmonoclonal anti-HIV-1, -HIV-2, -HTLV-1 or -HTLV-2 virus regulatoryprotein antibodies, characterized in that B cells originating fromindividuals immunized by an immunogenic compound according to thepresent invention are used, the said B cells being transformed by theEBV virus and producing specific anti-HIV-1, -HIV-2, -HTLV-1 or -HTLV2virus regulatory protein antibodies.

The above EBV+ cells can be cultured to produce the required antibodies.These cells, as has been seen, originate in particular from patientsimmunized with a native regulatory protein, or with an immunogeniccompound according to the invention.

The present Application also relates to a process for the preparation ofmonoclonal antibodies according to the invention to an HIV-1, HIV-2,HTLV-1 or HTLV-2 virus regulatory protein, which is inactivated ornon-inactivated, characterized in that hybridomas are prepared frommammals, in particular mice, from splenocytes or adenocytes, inparticular mice immunized with a native regulatory protein or animmunogenic compound according to the invention, and myeloma cells,preferably of line x63, by processes well-known in the prior art (Kohlerand Milstein).

The present Application also relates to a process for the production ofanti-HIV-1, -HIV-2, -HTLV-1 or -HTLV-2 virus regulatory proteinantibodies by the technology of genetic recombination, characterized inthat an immunogenic compound as defined above is used as the immunogen.

The present Application also relates to fragments F(ab')2 or Fab of thesaid antibodies; these can be obtained by enzymatic digestion, forexample.

The present invention furthermore relates to a process for passiveimmunization of subjects contaminated with the HIV virus using specificanti-HIV virus multiplication-regulating protein antibodies, andspecifically anti-Tat antibodies, which neutralize or block the harmfuleffects of this protein in its extracellular configuration and areprepared as indicated above, or fragments F(ab')2 or F(ab) of theseantibodies.

The present Application also relates to a process for active anti-HIV or-AIDS immunization using an immunogenic compound described above,combined with a constitutional (or structure) protein of an HIV virus,in particular the envelope glycoprotein gp 120 or gp 160 or a modifiedor non-modified peptide fragment thereof, or with an inactivated HIVvirus, or with an HIV virus depleted in its genomic RNA, for example byalkaline hydrolysis.

The invention also relates to a process for active anti-HIV or -AIDSimmunization using an immunogenic compound described above, combinedwith one or more immunogens based on inactivated cytokines, and inparticular interferon-α, TGF-β or TNF. In fact, as will seen below inthe experimental part, a combination of the effects of antibodiesaccording to the invention to regulatory proteins, and anti-interferon-αantibodies in particular, completely restores the immunosuppressioninduced by HIV.

The present Application thus also relates to a composition comprisingtwo immunogenic compounds, that is to say an immunogenic compounddescribed above and an immunogenic compound which is capable of inducingantibodies to a cytokine, such as interferon-α or TNF-α, as described,for example, in WO-A-9118454, and to a composition comprising anantibody to a cytokine, in particular interferon-α, and an aboveantibody to a regulatory protein.

The present Application also relates to a process for activeimmunization, characterized in that an immunogenic compound as definedabove, combined with a mineral, oily or synthetic immunity adjuvant, oralso an immunogenic compound as defined above, coupled or combined witha protein which increases its immunogenicity, is used as the immunogen.

These immunizations can be realized both curatively and preventively.

A derivative of the Tat protein is preferably used as the immunogen forall the processes above and below.

The present invention also relates to a process for hyperimmunization ofHIV-1, HIV-2, HTLV-1 or HTLV-2 seronegative or seropositive subjects,characterized in that an immunogen as defined above is used for theproduction of hyperimmune human sera, which can be intended inparticular for passive serotherapy by administration of purifiedspecific antibodies or their fragments (F(ab')2 or Fab.

The invention also relates to a pharmaceutical composition comprising atleast one anti-virus regulatory protein antibody as defined above orobtained by the above processes as the curative or preventive activeprinciple.

The invention finally relates to an immunogenic compound or an aboveantibody for the preparation of a medicament intended for treatment ofthe harmful effects of an HIV-1, HIV-2, HTLV-1 or HTLV-2 virusregulatory protein.

In summary, and in particular, the present invention relates to thepreventive or curative use on a seropositive subject or a subjectsuffering from ARC/AIDS of specific antibodies to block the harmfulaction of an HIV-1, HIV-2, HTLV-1 or HTLV-2 virus regulatory protein,and in particular circulating Tat. These specific antibodies mayoriginate: 1. from the subject himself, induced by an activeimmunization (vaccination) with biologically inactivated but immunogenicTat, called Tat-toxoid by analogy to bacterial toxoids, or 2. from aforeign allo- or xenogenic organism, administered to the subject bypassive immunization (serotherapy). The allogenic antibodies (in humans)may be generated in volunteer non-infected subjects after activeimmunization (vaccination) with a regulatory protein, such as Tat or itsderivatives (Tat-toxoid or peptide fragments of Tat according to theinvention). These passively administered antibodies, whether allogenic(human) or xenogenic (animal), can be complete monoclonal or polyclonalantibodies or fragments F(ab')2 or Fab of antibodies.

Tat-toxoid, as has been indicated above, is understood as meaning apeptide or a Tat protein treated with a chemical agent. This treatmenthas caused the toxic biological properties of circulating Tat(immunosuppression of T cells; induction of the production ofinterferon-α by cells which produce interferon; neoangiogenesis ofendothelial cells; blockage of the antiviral effect of interferon on themacrophages) to be lost to the molecule, but has retained the propertiescapable of inducing the formation of antibodies, when presented andprepared in an appropriate manner, coupled or not to a "carrier",aggregated or not, in the presence or absence of an adjuvant.

The concept of Tat-toxoid has been extended to immunogenic peptidefragments of Tat, that is to say a peptide sequence of Tat which iscapable of inducing the formation of anti-Tat antibodies when presentedin an appropriate manner, coupled or not to a carrier, in the presenceor absence of an adjuvant.

The invention also relates to pharmaceutical compositions.

a) A pharmaceutical composition comprising a viral toxoid or a fragmentor analogue of regulatory protein, in particular Tat, according to theinvention as the preventive or curative agent.

b) A pharmaceutical composition comprising anti-Tat antibodies producedfrom organisms immunized against the said protein or its fragmentsF(ab')2 or Fab, according to the invention, as the preventive orcurative agent.

The invention also proposes a kit comprising a vaccine pharmaceuticalcomposition which, in addition to the active principle (Tat-toxoid orits derivatives or anti-Tat antibodies) can comprise an adjuvant and/oranother immunogen having anti-retrovirus properties.

Finally, the invention proposes a pharmaceutical composition in aconventional galenical form. In particular, the active principleaccording to the invention is combined, in an amount sufficient to beeffective from a therapeutic point of view, with a diluent or a carrierwhich is acceptable from a pharmaceutical point of view.

Experiment 1 Pathogenic Effects of Circulating Tat Protein

Immunosuppression of T cells in the presence of Tat:

The effect of Tat on activated T lymphocytes of peripheral blood hasbeen investigated. Mononuclear cells (PMBC) and T lymphocytes (HLA 35DR-) isolated by immunomagnetism were activated by anti-CD₃ antibodiesin the presence or in the absence of the Tat molecule. After culture for5 days, the cell proliferation was measured by incorporation of H₃-thymidine. The results showed that Tat inhibited the proliferation ofHLA DR- T cells (85% inhibition at a concentration of 1.5 μg per ml).This inhibition disappeared in the presence of specific anti-Tatantibodies (Intracel, U.K.).

Experiment 2 Pathogenic Effects of Circulating Tat Protein

Inhibition of the effect of interferon on macrophages in the presence ofTat:

The effect of Tat on the antiviral action of interferon-α was measuredby the conventional biological test using measurement of thecytopathogenic potency of the VSV virus on MDBK cells.

Effect of Tat on exogenous interferon

Increasing doses of Tat enabled the anti-viral potency of exogenousinterferon to be inhibited from low concentrations according to adose/effect curve for concentrations of Tat ranging from 1 to 20 μg/ml.In a representative experiment, the protective effect of interferon-αapparent up to the dilution 1/4,800 (corresponding to 150 I.U.) isreduced to the dilution 1/300 for concentrations of Tat of 10 μg/ml. Atthe concentration of 10 μg/ml of Tat, at the dilution of 4,800 ofexogenous interferon the titre of VSV is thus increased from 10³.8(interferon samples without Tat) to 10⁵.5 (interferon samples in thepresence of Tat). In these experiments, the inhibition of the anti-viraleffect of interferon-α by the Tat molecule is suppressed in the presenceof specific anti-Tat antibodies (Intracel, U.K.), and also by the horseantibodies which we prepared (see examples below).

Experiment 3 Detection of the Tat Protein in its ExtracellularConfiguration in Subjects Infected by HIV-1

Presence of anti-Tat antibodies in the serum of seropositive patients:

a) An ELISA study of serum anti-Tat antibodies using the native Tatmolecule as the antigen was carried out on 50 seropositive subjects and15 seronegative subjects. All the sera of the seronegative subjects and20 sera of the seropositive subjects revealed no presence of anti-Tatantibodies with an optical density below the threshold (cut-off)(OD=0.250). Among the 30 sera of subjects infected with HIV whichexceeded the threshold, 6 have an OD greater than 0.500. No apparentcorrelation could be made between the presence of anti-Tat antibodies onthe one hand, and the clinical condition and the number of CD4 cells permm³ of blood on the other hand.

b) An ELISA study of serum anti-Tat antibodies using various peptides ofTat as antigens was carried out on seropositive subjects at variousstages of evolution of their HIV infection--asymptomatic subjects;patients showing pre-AIDS clinical symptoms (ARC) and patients sufferingfrom AIDS. The results of this study are as follows:

1) The peptide sequences of Tat

Met Glu Pro Val Asp Pro Arg Leu Glu Pro Trp Lys His Pro Gly (SEQ IDNO:2) (residues 1-15 in the N terminal)

His Gln Val Ser Leu Ser Lys Gln Pro Thr Ser Gln Pro Arg Gly Asp (SEQ IDNO:1) (residues 65-80 in the C terminal)

were recognized by the great majority of sera of seropositive subjectsand by no serum of seronegative subjects (controls). The reactions werevery positive, but no correlation could be demonstrated with respect tothe evolution of the HIV infection and the number of CD4 in thesubjects.

2) The other sequences studied were not recognized in an appreciablemanner by the sera, whether from seropositive individuals or controls(see Table 1). Only a few rare sera for each sequence showed an opticaldensity greater than the threshold (twice the mean of the control sera).

                  TABLE 1                                                         ______________________________________                                                  Infected subjects*                                                                             Control subjects                                     (72 sera)  (10 sera)                                                        Peptide     Positive                                                                              Mean       Positive                                                                            Mean                                       residues of Tat sera** OD sera** OD                                         ______________________________________                                         1-15       67      0.43       0     0.08                                        9-20 0 0.26 0                                                                22-37 7 0.78 0 0.41                                                           36-50 5 0.86 0 0.65                                                           46-60 6 0.53 0 0.40                                                           52-60 2 0.32 0 0.38                                                           56-70 0 0.19 0 0.20                                                           65-80 70 1.01 0 0.08                                                        ______________________________________                                         *The subjects infected by HIV are at various stages; asymptomatic             subjects, patients suffering from ARC and patients suffering from AIDS.       **The reaction is considered positive (threshold) if the optical density      is twice as high as the mean of the seronegative subjects.               

3) It is interesting to note that the most reactive sequence (AA 65-80)is that containing the RGD residues recognized by the integrins.

EXAMPLE 1 Preparation of Immunogenic Tat Protein (Tat-toxoid)

Inactivation of the Tat protein in the presence of formaldehyde

Formaldehyde is added to a solution of Tat (1 mg/ml) in 70 mM disodiumphosphate, pH 8.0, to the final concentration of 33 mM.

The mixture is incubated at 37° C. for 1, 3, 5, 7 and 9 days. At the endof each of the incubation periods, a sample is taken and the reactionwith formaldehyde is blocked by addition of glycine to the finalconcentration of 100 mM.

Each sample is dialysed for 1 night at 40° C. against 100 times itsvolume of PBS (phosphate-buffered saline).

EXAMPLE 2 Preparation of Immunogenic Tat Protein (Tat-toxoid)

Inactivation of the Tat protein in the presence of glutaraldehyde

Glutaraldehyde is added to a solution of Tat (1 mg/ml) in 70 mM disodiumphosphate, pH 8.2, to the final concentration of 0.026 M or 0.0026 M.The reaction with the glutaraldehyde is allowed to continue for varioustimes varying from 1 min to 3 h. After the reaction time chosen, at thelaboratory temperature, the reaction is blocked by addition of glycineto the final concentration of 100 mM. The various samples are dialysedfor 16 hours at 4° C. against 100 times their volume of PBS.

EXAMPLE 3 Preparation of Immunogenic Tat Protein (Tat-toxoid)

Inactivation and simultaneous coupling to tetanic toxin of Tat

Glutaraldehyde is added to a mixture of tetanic toxin and Tat in a molarratio of 1 to 15 in a 70 mM-100 mM phosphate buffer (pH varies from 6.8to 8.2) to the final concentration (varying from 0.0026 M to 0.026 M),and the inactivation and coupling reaction is allowed to take place forvarious times (3-15 min) at the laboratory temperature. The reaction isblocked by addition of glycine to the final concentration of 100 mM andthe various samples are dialysed for 16 hours at 4° C. against 100 timestheir volume of PBS.

EXAMPLE 4 Immunogenic Potency and Antigenicity of Tat-toxoids

Immunogenic Tat-toxoid in the mouse: Antibodies by ELISA

The immunogenic potency of various preparations of Tat which has beeninactivated, either by formaldehyde (Tat-toxoid), or by glutaraldehyde(Tat-polan), or after coupling to tetanic toxin (conjugated Tat-tetanictoxin), that is to say the immunogenic compounds of examples 1 to 3, wasdetermined in the mouse.

Swiss mice weighing 18-20 g were immunized by 2 subcutaneous injectionsof the immunogen of examples 1, 2 and 3 in the presence of Freund'scomplete adjuvant, the second being carried out 3 weeks after the 1stwith 20 μg of preparation in emulsion in the presence of Freund'sincomplete adjuvant.

15 days after the repeat injection, blood samples were takenintracardially. The anti-Tat antibodies in the sera were determined byELISA. The optical density was measured at 490 nm.

The results obtained, summarized in Table 2, show that all the Tatpreparations are immunogenic to various degrees, except for theconjugate Tat-TT treated with glutaraldehyde for a short time (3minutes), which proved to be toxic and killed the mice within 24 hours(TT to weakly inactivated). It should be pointed out that the sera ofthe non-immunized mice respond below 0.200 (OD) to native Tat in thesame way as to Tat-toxoid. Furthermore, these results show that thenative Tat is recognized by the antibodies in the same way as thetoxoid, which confirms their equivalent antigenicity and also justifiesthe use of the Tat-toxoid for immunization.

                  TABLE 2                                                         ______________________________________                                                           Level of anti-                                                                           Level of anti-                                     native Tat Tat-toxoid                                                         antibodies opti- antibodies (3                                                cal density days) optical                                                    Preparations (OD) density (OD)                                              ______________________________________                                        Tat-toxoid (1 day) 0.320      0.331                                             Tat-toxoid (3 days) 0.465 0.494                                               Tat-toxoid (5 days) 0.998 0.901                                               Tat-toxoid (9 days) 0.807 0.780                                               Tat-polan (1 min; 0.026 M) 0.718 0.706                                        Tat-polan (15 min; 0.026 M) 1.564 1.452                                       Tat-polan (60 min; 0.0026 M) 1.065 1.113                                      Tat-polan (3 h; 0.0026 M) 0.194 0.210                                         Conjugated Tat-TT (3 min; 0.0026 M) toxic toxic                               Conjugated Tat-TT (15 min; 0.0026 M) 1.987 1.897                              Conjugated Tat-TT (6 min; 0.026 M) 0.824 0.795                                Conjugated Tat-TT (15 min; 0.026 M) 1.642 1.556                             ______________________________________                                    

EXAMPLE 5 Acute Toxicity

The toxicity of preparations of Tat-toxoid and Tat-polan was determinedby subcutaneous injection to Swiss mice weighing 18-20 g. Thepreparations of Tat-toxoid (7 days) and Tat-polan (15 min; 0.026 M) wereadministered at a dose of 100 μg per mouse. The animals were observedfor 7 days.

No manifest sign of toxicity was observed. The weight of the animalscontinued to increase. Macroscopic examination of the organs on autopsyrevealed no anomalies.

EXAMPLE 6 Anti-Tat Antibodies

Anti-Tat antibodies were prepared as follows: The IgG fraction wasisolated over a column of G protein from sera of mice immunized withTat-polan. The fragments F(ab')2 were also prepared from the IgGfraction isolated by peptic digestion. The immunological reactivity ofthese fractions was checked by ELISA.

EXAMPLE 7 Biological Inactivity of Tat-toxoid

The various Tat-toxoids inactivated for 1, 3, 5 and 9 days from example1 were tested in a test of the expression activity of the gene reporterchloramphenicol acetyl transferase (CAT) dependants of "long terminalrepeat" (LTR) of HIV-1.

The principle of this test is as follows: Cells of the HeLa linecontaining the CAT gene under control of LTR of HIV are brought intocontact with native Tat or the toxoids for 6 h. After culture for 24 h,the cells are lysed and the amount of CAT produced is measured by a testfor the activity of CAT, which measures the percentage of acetyl-CoAattached to chloramphenicol. If the Tat molecule is active, there willbe high percentage of acetylated chloramphenicol, and if not thepercentage will be low. Culture of these adherent cells is carried outin a standard manner in RPMI, 10% FCS medium. The concentration of Tatadded to give an effect/dose ranges from 1 μg to 10 μg per ml ofsupernatant.

The results shown here demonstrate that the Tat-toxoid is inactivatedtotally after 3 days, with a low residual activity at 1 day.

    ______________________________________                                        Preparations Acetylation of chloramphenicol (%)                               ______________________________________                                        Native Tat   100                                                                Tat-toxoid (1 day) 18                                                         Tat-toxoid (3 days) 3                                                         Tat-toxoid (5 days) 2                                                         Tat-toxoid (9 days) 3                                                       ______________________________________                                    

EXAMPLE 8 Absence of Pathogenic Effects of Tat-toxoid

a) Absence of immunosuppression of T cells in the presence of Tat-toxoid(in contrast to experiments 1 and 2).

The effect of Tat-toxoid (5 days) on activated T lymphocytes ofperipheral blood has been investigated. Mononuclear cells (PMBC) and Tlymphocytes (HLA 35 DR-) isolated by immunomagnetism were activated byanti-CD₃ antibodies in the presence or absence of immunogenic compoundsof example 1. After culture for 5 days, the cell proliferation wasmeasured by incorporation of H₃ -thymidine. The results showed that, incontrast to native Tat, the Tat-toxoid did not inhibit the proliferationof HLA DR- T cells. Thus, at a dose of 5 μg/ml in the culture in thepresence of Tat-toxoid, the proliferation of cells was identical to thatof the control.

b) Absence of inhibition of the effect of interferon-α on macrophages inthe presence of Tat-toxoid:

The effect of Tat-toxoid on the antiviral action of interferon-α wasmeasured by the conventional biological test using measurement of thecytopathogenic potency of the VSV virus on MDBK cells.

Effect of Tat-toxoid on exogenous interferon

In contrast to native Tat, equivalent doses of Tat-toxoid of example 1did not enable the anti-viral potency of exogenous interferon to beinhibited. In a representative experiment, the protective effect ofinterferon-α apparent up to the dilution 1/4,800 (corresponding to 150I.U.) is maintained in the presence of 50 μg of Tat-toxoid, while it isreduced to 1/300 with native Tat. At the dilution 4,800 of exogenousinterferon, the titre of VSV is maintained at the level of the controlat 10³.8 (interferon samples with Tat-toxoid), while it increases to10⁵.5 (interferon samples in the presence of Tat).

EXAMPLE 9 Protective Role of Anti-Tat-toxoid Antibodies with Regard tothe Effects of Native Tat

Antibodies to Tat-toxoids of experiment 1 (treatment with formaldehydefor 3 days) were prepared in hyperimmunized horses. From theseantibodies, fragments F(ab')2 were also prepared in accordance with theprocedure described in example 6.

The antibodies, like the fragments F(ab')2, inhibit the variousbiological activities of native Tat in the various tests:Transactivation of the LTR of HIV (CAT test) (see example 7),immunosuppression (see experiment 1), inhibition of the effect ofexogenous interferon-α on the culture (see experiment 2). Experiments 1and 2 and example 7 were repeated with native Tat with or withoutpreincubation of this native Tat for 1 h at 37° with the antibodies orfragments F(ab')2 at a dose of 40 μg of antibodies per 1 μg of nativeTat. The results showed that the antibodies inhibit the action of nativeTat.

EXAMPLE 10 Systematic Recovery of Cell Proliferation Due to theCombination of Anti-Tat Antibodies and Anti-interferon-α Antibodies:Synergism of Anti-Tat and Anti-interferon-α Antibodies

It is found that PBLs (mononuclear cells of the peripheral blood) ofhealthy subjects infected in vitro with HIV-1 and cultured for 6 dayshad an immunosuppressive activity.

In fact, if such cells infected for 6 days (or non-infected) andirradiated are added in a proportion of 1 to 5 to autologous cellsactivated by the protein Staphylococcus enterotoxin B (SEB), it is foundthat after 4 days the proliferation of the autologous cells is reducedby 80% with respect to the controls (non-infected cells).

In this model, if anti-interferon-α antibodies are added to the cultureof the cells infected in vitro, these cells then lose their suppressoreffect in 50% of subjects. In 20% of the cases, suppressive cells lose60% of their suppressor effect, and in 30% this suppressor effect ismaintained significantly.

EXAMPLE 11 Preparation of Immunogenic Nef Protein (Nef Toxoid)

Inactivation of the Nef protein in the presence of formaldehyde.

Formaldehyde is added to a solution of Nef protein (1 g/ml) in 70 mMdisodium phosphate, pH 8.0, to the final concentration of 33 mM.

The mixture is incubated at 37° C. for 1, 3, 5, 7 and 9 days. At the endof each of the incubation periods, a sample is taken and the reactionwith formaldehyde is blocked by addition of glycine to the finalconcentration of 100 mM.

Each sample is dialysed for 1 night at 4° C. against 100 times itsvolume of PBS (phosphate-buffered saline), and the Nef-toxoid isisolated.

EXAMPLE 12 Preparation of Immunogenic Nef Protein (Nef-polan)

Inactivation of the Nef protein in the presence of glutaraldehyde.

Glutaraldehyde is added to a solution of Nef (1 mg/ml) in 70 mM disodiumphosphate, pH 8.2, to the final concentration of either 0.026 M or0.0026 M. The reaction with the glutaraldehyde is allowed to continuefor various times varying from 1 min to 3 h. After the reaction timechosen, at the laboratory temperature, the reaction is blocked byaddition of glycine to the final concentration of 100 mM. The varioussamples are dialysed for 16 hours at 4° C. against 100 times theirvolume of PBS and the Nef-polan is isolated.

EXAMPLE 13 Immunogenic Potency and Antigenicity of Nef-toxoids

Immunogenic Nef-toxoid in the mouse: Antibodies by ELISA.

The immunogenic potency of various preparations of Nef which has beeninactivated, either by formaldehyde (Nef-toxoid) or by glutaraldehyde(Nef-polan), that is to say the immunogenic compounds of examples 11 and12, was determined in the mouse.

Swiss mice weighing 18-20 g were immunized by 2 subcutaneous injectionsof the immunogen of examples 12 and 13 in the presence of Freund'sadjuvant, the second being carried out 3 weeks after the first with 20μg of preparation in emulsion in the presence of Freund's incompleteadjuvant.

15 days after the repeat injection, blood samples were takenintracardially. The anti-Nef antibodies in the sera were determined byELISA. The optical density was measured at 490 nm.

The results obtained, summarized in the table below, show that all theNef preparations are immunogenic to various degrees. It should bepointed out that the sera of non-immunized mice give a level of responselower than 0.2 (in OD) in these ELISA tests.

These results show that the native Nef protein is recognized by theantibodies in the same way as the toxoid, which confirms theirequivalent antigenicity and also justifies the use of the Nef-toxoid forimmunization.

    ______________________________________                                                        Level of anti-                                                                            Level of anti-Nef-                                   native Nef toxoid antibodies                                                  antibodies op- (3 days) opti-                                                Preparations tical density (OD) cal density (OD)                            ______________________________________                                        Nef-toxoid (1 day)                                                                            0.56        0.5                                                 Nef-toxoid (3 days) 0.78 0.8                                                  Nef-toxoid (5 days) 0.99 1.01                                                 Nef-toxoid (9 days) 0.65 0.75                                                 Nef-polan (1 min; 0.026 M) 1.12 0.98                                          Nef-polan (15 min; 0.026 M) 1.69 1.56                                         Nef-polan (60 min; 0.0026 M) 1.31 1.41                                        Nef-polan (3 h; 0.0026 M) 0.35 0.41                                         ______________________________________                                    

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 2                                           - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - His Gln Val Ser Leu Ser Lys Gln Pro Thr Se - #r Gln Pro Arg Gly        Asp                                                                             1               5   - #                10  - #                15              - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - Met Glu Pro Val Asp Pro Arg Leu Glu Pro Tr - #p Lys His Pro Gly          1               5   - #                10  - #                15             __________________________________________________________________________

What is claimed is:
 1. An isolated modified protein comprising aderivative of a native HIV-1, HIV-2, HTLV-1 or HTLV-2 viral regulatoryprotein, which derivative comprises all or a fragment of the nativeregulatory protein having modifications comprising deletions,substitutions or additions of the amino acid residues thereof, and/orhaving chemical functionalization of the amino acid residues thereof byacylation or treatment with an aldehyde or with a coupling agent, saidmodifications and/or functionalizations causing said modified protein tolose at least 50% of the toxic biological properties of thecorresponding said native viral regulatory protein and yet to retain thecapability of generating polyclonal antibodies cross-reactive with thecorresponding said native viral regulatory protein, wherein less than30% of the amino acids have been modified, with the proviso that thefunctionalization does not cause conjugation to a carrier protein.
 2. Amodified protein in accordance with claim 1, wherein said derivativecomprises said native protein or fragment thereof having chemicalfunctionalization of the amino acid residues thereof by acylation ortreatment with an aldehyde or with a coupling agent.
 3. A process forpreparing a modified protein in accordance with claim 2,comprising:chemically treating said native protein or fragment with analdehyde or with a coupling agent, or to acylate amino acid residuesthereof; and purifying the treated native protein or fragment.
 4. Aprocess in accordance with claim 3, wherein said chemically treatingcomprises chemically treating said native protein or fragment with analdehyde.
 5. The process in accordance with claim 4, wherein saidaldehyde is formaldehyde.
 6. The process in accordance with claim 4,further comprising treating the treated native protein or fragment withirradiation.
 7. The process in accordance with claim 6, wherein theirradiation is UV irradiation.
 8. A modified protein in accordance withclaim 2, wherein said derivative comprises said native protein orfragment thereof having chemical functionalization of the amino acidresidues thereof by acylation.
 9. A modified protein in accordance withclaim 2, wherein said derivative comprises said native protein orfragment thereof having chemical functionalization of the amino acidresidues thereof by treatment with a coupling agent so as to form anaggregate of the native protein.
 10. A modified protein in accordancewith claim 9, wherein said coupling agent is glutaraldehyde.
 11. Amodified protein in accordance with claim 2, wherein said derivativecomprises said native protein or fragment thereof having chemicalfunctionalization of the amino acid residues thereof by treatment withan aldehyde.
 12. A modified protein in accordance with claim 11, whereinsaid aldehyde is formaldehyde.
 13. A modified protein in accordance withclaim 1, wherein said derivative comprises said native protein orfragment thereof having modifications comprising deletions,substitutions or additions of the amino acid residues thereof, whereinless than 30% of the amino acids have been modified.
 14. A modifiedprotein in accordance with claim 13, wherein less than 20% of the aminoacids have been modified.
 15. A modified protein in accordance withclaim 13, wherein less than 10% of the amino acids have been modified.16. A modified protein in accordance with claim 1, wherein saidderivative is of a full length native HIV-1, HIV-2, HTLV-1 or HTLV-2viral regulatory protein.
 17. A modified protein in accordance withclaim 1, wherein said derivative is of a fragment of a native HIV-1,HIV-2, HTLV-1 or HTLV-2 viral regulatory protein.
 18. A modified proteinin accordance with claim 17, wherein said derivative comprises saidfragment having chemical functionalization of the amino acid residuesthereof by treatment with an aldehyde.
 19. A modified proteincomposition in accordance with claim 1, wherein said native protein is aviral regulatory protein selected from the group consisting of the Nef,Tat, Vif and Rev regulatory protein of HIV-1 or HIV-2.
 20. A modifiedprotein in accordance with claim 19, wherein said viral regulatoryprotein is Tat.
 21. The modified protein in accordance with claim 1,wherein said modified protein is the Tax regulatory protein of HTLV-1 orHTLV-2.
 22. The modified protein in accordance with claim 1, wherein thetoxic biological properties are immunosuppression of T cells,deregulation of interferon-α production, or neoangiogenesis.
 23. Themodified protein in accordance with claim 1, wherein the modifiedprotein has lost at least 80% of the toxic biological properties of saidnative viral regulatory protein.
 24. The modified protein in accordancewith claim 1, wherein the modified protein has lost at least 95% of thetoxic biological properties of said native viral regulatory protein. 25.An immunogenic composition, comprising, as active ingredient, aderivative of a native HIV-1, HIV-2, HTLV-1 or HTLV-2 viral regulatoryprotein, which derivative comprises all or a fragment of the nativeregulatory protein, having modifications comprising deletions,substitutions or additions of the amino acid residues thereof, and/orhaving chemical functionalization of the amino acid residues thereof,said modifications and/or functionalizations causing said modifiedprotein to lose at least 50% of the toxic biological properties of saidnative viral regulatory protein and yet to retain the capability ofgenerating polyclonal antibodies cross-reactive with said native viralregulatory protein, wherein less than 30% of the amino acids have beenmodified, with the proviso that the functionalization does not causeconjugation to a carrier protein, and a pharmaceutically acceptableadjuvant.
 26. The immunogenic composition in accordance with claim 1,further comprising an immunogenic compound in association with themodified protein.
 27. The immunogenic composition in accordance withclaim 26, wherein said immunogenic compound is selected from the groupconsisting of tetanic toxoid, gp120 or gp160 protein which is native orinactivated by physical, chemical, genetic or immunological treatment,and an immunogenic fragment of said gp120/gp160 protein.